This invention relates to crystallography and immunology, and, in particular, to the elucidation, for the first time, of the three-dimensional structure of the Fabxe2x80x2 fragment of monoclonal antibody 2F5.
The monoclonal antibody (Mab) 2F5 is a potent neutralizer of both laboratory strains and primary isolates of most clades of HIV-1, reacting with the largely conserved peptide sequence ELDKWAS (SEQ ID No: 1) of the virus envelope protein gp41, sometimes called the Katinger Epitope (refs. 1 to 7. Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). As such, Mab 2F5 is of major interest in the development of an HIV-1 vaccine. Based on studies of immunogenic presentation, the antigenicity of the epitope sequence was concluded to be contingent upon its molecular context (ref. 8).
In accordance with the present invention, there is provided the three-dimensional structure of the Fabxe2x80x2 fragment of Mab 2F5, both uncomplexed and with bound epitope. In the complexed crystalline structure, the seven amino acid sequence (ELDKWAS; SEQ ID No: 1) forms a slightly distorted xcex2 turn, with the central DKW core accounting for the majority of protein/peptide interactions, as discussed below.
As can be seen from the detailed analysis provided herein, the slightly-distorted xcex2 turn is stabilized by hydrogen bonds from aspartate backbone and sidechain to alanine and tryptophan amides respectively. In the three-dimensional structure, tryptophan and lysine sidechains of the epitope are stacked and parallel.
The elucidation of these three-dimensional structures enables there to be constructed, as set forth herein, peptide-mimetics constrained in the same xcex2-turn-like configuration as seen in the crystal structure of the complex, which would be expected to increase the immunogenicity of the epitope sequence.
Accordingly, in one aspect of the invention, there is provided an isolated crystal of the Fabxe2x80x2 fragment of monoclonal antibody 2F5. The isolation of the crystalline form of the Fabxe2x80x22F5 fragment enables the three-dimensional structure of such form of the fragment to be determined and such structure is shown in FIG. 1, described below. Certain characterizing parameters have been determined for the crystal structure, as set forth in Table 2 below.
The isolated crystal may be grown in space group P212121 with cell dimensions a=63.6 xc3x85; b=76.4 xc3x85; c=93.4 xc3x85, although the crystals may be grown in another space group with its own unique cell dimensions. The crystalline form of the Fabxe2x80x22F5 may have the atomic coordinates deposited on Apr. 9, 1999 with the Protein Data Bank under Accession No. 2F5A.
Fabxe2x80x22F5 molecules organized in the isolated crystal provided herein possess a third hypervariable (V3) loop of the heavy chain comprising amino acid residues H98 to H120, as seen in Table 1 below, which has a three-dimensional structure as shown in FIG. 4, described below and atomic coordinates as shown in Table 3 below.
In accordance with a further aspect of the present invention, there is provided an isolated crystal of the Fabxe2x80x2 fragment of monoclonal antibody 2F5 complexed with a peptide having the amino acid sequence ELDKWAS (SEQ ID No: 1) or a functional analog thereof. The solution of the crystal form of the complex enables the three-dimensional structure of such form of the complex to be determined and the detail of the binding site of the peptide to the Fabxe2x80x2 fragment is shown in FIG. 3, described below. Certain characterizing parameters have been determined for the crystal structure of the complex, as set forth in Table 2 below.
The isolated crystal complex may be grown in space group P212121 with cell dimensions a=58.0 xc3x85; b=65.0 xc3x85; c=175.6 xc3x85, although the crystal complex may be grown in another space group with its own unique cell dimensions. The crystalline form of the complexed form of the Fabxe2x80x22F5 may have the atomic coordinates deposited with the Protein Data Bank under Accession No. 2F5B on Apr. 9, 1999.
The functional analog of the amino acid sequence ELDKWAS may be one in which lysine is replaced by arginine and/or one in which tryptophan is replaced by tyrosine, phenylalanine or uncharged histadine. One example of such functional analog is ELDRWAS (SEQ ID No: 2).
The elucidation of the crystal structure of the Fabxe2x80x22F5 fragment when bound to the peptide ELDKWAS (SEQ ID No: 1), provides details of the actual conformation of the peptide epitope when it is bound to the antibody, which will be the same, irrespective of the kind of crystal which is analyzed.
The information which is provided concerning the conformation of peptide epitope then provides the basis for the provision of peptide analogs, peptide mimetics and other antigens which are potentially useful as components of an anti-HIV vaccine.
Accordingly, in another aspect of the present invention, there is provided a synthetic peptide which binds to monoclonal antibody 2F5 and which is constrained to provide a three-dimensional structure corresponding to that for the peptide ELDKWAS (SEQ ID No: 1) shown in FIG. 3.
This synthetic peptide may contain the amino acid sequence DKW or a functional analog thereof and may be constrained in the slightly distorted xcex2-turn configuration of the three-dimensional structures with the tryptophan and lysine residue chains stacked and parallel, as seen in FIG. 3 and as discussed in more detail below.
The analysis of the three-dimensioned conformation of the epitope indicates that at least one of the tryptophan and lysine sidechains may be substituted by an amino acid which retains the peptide-protein. interaction shown in FIG. 3, which also binds to the Mab. For example, arginine (R) may be used in place of lysine (K) and tyrosine (Y), phenylalanine (F) and uncharged histadine (H) may be used in place of tryptophan (W). Peptides wherein one or more of such amino acid substitution is effected are peptides which contain a xe2x80x9cfunctional analogxe2x80x9d of the amino acid sequence DKW, as the term is understood herein, in that the peptide still bind to the monoclonal antibody 2F5.
The synthetic peptide provided herein may be constrained in the required conformation by any convenient means. For example, a disulphide bridge may be used to maintain the amino acid sequence DKW or analogs thereof in the respective orientation of two amino acid residues as shown in FIG. 3. Such disulphide bridge may be provided between cysteine residues in the synthetic peptide ECDKWCS (SEQ ID No.: 3).
Alternatively, a lactam bond may be used to maintain the amino acid sequence DKW or functional analogs thereof in the respective orientation of the amino acid residues as shown in FIG. 3. Such lactam bond may be formed between diaminopropionic acid (Dap) and glutamate (E) residues in the synthetic peptide EdapDKWES (SEQ ID No.: 4) or EEDKWDapS (SEQ ID No.: 5).
It is well known that the immunogenicity of peptides may be enhanced by conjugation to carrier molecules, such as protein, including diphtheria toxoid, tetanus toxoid or an outer membrane protein of Haemophilus. Such carrier protein may be linked to the peptide.
There is also provided, in an additional aspect of the invention, a method of making a peptide binding to monoclonal antibody 2F5, which comprises co-crystallizing a Fabxe2x80x2 fragment of the monoclonal antibody 2F5 with a peptide having the amino acid sequence ELDKWAS (SEQ ID No.: 1) or functional analog thereof to form a crystalline complex; analyzing the crystalline complex to determine the three-dimensional orientation of the bound peptide in relation to the Fabxe2x80x2 fragment; and synthesizing a peptide containing at least amino acids DKW or functional analogs thereof constrained in the determined three-dimensioned orientation.
The functional analog of the peptide containing at least amino acids DKW is one which still binds to the monoclonal antibody 2F5. Functional analogs also extend to known analogs of the ELDKWAS motif, including those of the formula X1LDKWX2S wherein Xl is E, A, G or Q and X2 is A or T.